EP1055144B1 - Systeme optique a element spectroselectif - Google Patents

Systeme optique a element spectroselectif Download PDF

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Publication number
EP1055144B1
EP1055144B1 EP99915483.4A EP99915483A EP1055144B1 EP 1055144 B1 EP1055144 B1 EP 1055144B1 EP 99915483 A EP99915483 A EP 99915483A EP 1055144 B1 EP1055144 B1 EP 1055144B1
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European Patent Office
Prior art keywords
laser scanning
scanning microscope
spectrally selective
confocal laser
selective element
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Expired - Lifetime
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EP99915483.4A
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German (de)
English (en)
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EP1055144A1 (fr
Inventor
Johann Engelhardt
Joachim Bradl
Heinrich Ulrich
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Leica Microsystems CMS GmbH
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Leica Microsystems CMS GmbH
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders

Definitions

  • the invention relates to a confocal laser scanning microscope with an optical arrangement which is arranged in the beam path of a light source suitable for fluorescence excitation and in the beam path of the confocal laser scanning microscope, wherein the optical arrangement at least one spectrally selective element for coupling the excitation light at least a light source in the microscope and to hide the scattered at the object and reflected excitation light or the excitation wavelength from the light coming from the object via the detection beam path.
  • color beam splitters with a very special transmission and reflection characteristic are used in the beam path of a light source suitable for fluorescence excitation. Most of these are dichroic beam splitters. With such an element, the fluorescence excitation wavelength ⁇ ill1 (or ⁇ ill2 , ⁇ ill3 ..., ⁇ illn when using multiple lasers) is reflected in the illumination beam path to excite the fluorescence distribution in the object and then along with the scattered at the object reflected excitation light to pass through the beam path up to the color beam splitter.
  • the excitation light having the wavelengths ⁇ ill1 , ⁇ ill2 , ⁇ ill3 ,..., ⁇ illn is reflected back to the laser beam at the color beam splitter , namely out of the detection beam path .
  • the fluorescent light with the wavelengths ⁇ fluo1 , ⁇ fluo2 , ⁇ fluo3 , ...., ⁇ fluon passes through the color beam splitter and is detected - possibly after further spectral splitting.
  • Color beam splitters are usually realized by an interference filter and are selectively vaporized depending on the wavelengths used for the excitation or for the detection.
  • color beam splitters In practice, the use of color beam splitters is initially disadvantageous insofar as this involves expensive and therefore expensive optical components in the production. Furthermore, it is disadvantageous that color beam splitters have a fixed wavelength characteristic and therefore can not be used with any flexibility with regard to the wavelength of the excitation light. When changing the wavelength of the excitation light and the color beam splitter must be replaced, such as in an arrangement of multiple color beam splitter in a filter wheel. This is again consuming and therefore expensive and, moreover, requires a very special adjustment of the individual color beam splitter.
  • a color beam splitter entails the further disadvantage that light losses caused by reflection occur, in particular light losses of fluorescent light which is just to be detected.
  • the spectral transmission / reflection range is quite wide in the case of color beam splitters ( ⁇ ill ⁇ 20 nm) and by no means ideally "steep". Consequently, the fluorescent light from this spectral range can not be detected ideally.
  • the number of simultaneously injectable laser is limited, namely, for example, on the number of arranged in a filter wheel and combinable color beam splitter.
  • a maximum of three lasers are coupled into the beam path.
  • all color beam splitters, including the color beam splitters arranged in a filter wheel have to be precisely adjusted, which involves a considerable outlay in terms of handling.
  • suitable neutral beam splitters can be used which efficiently guide the fluorescent light to the detector together with the excitation light scattered / reflected by the object.
  • the losses in the laser coupling are significant.
  • a luminescent system for examining skin tissue is known. This system is used to chemically analyze the tissue, producing no image of the tissue. This is done by first illuminating the sample to be examined with excitation light for a certain period of time and then, with the illumination switched off, detecting the luminescence light emanating from the sample and having a different wavelength than the excitation light. Excitation and detection alternate in time.
  • a spectral detector which can also be used in a confocal fluorescence microscope.
  • a beam splitter For coupling of illumination light is a beam splitter.
  • the present invention is based on the object, a confocal laser scanning microscope to design and further develop that the Coupling of the excitation light of different excitation wavelengths is possible without having to make a change or a special adjustment of the optical elements used there when changing the wavelength of the excitation light. Furthermore, the number of required optical elements should be reduced as much as possible. Finally, an ideal detection of the fluorescent light should be possible.
  • the spectrally selective element is adjustable to the excitation wavelength trainees.
  • excitation light of different wavelengths can be masked out and correspondingly coupled in by the spectrally selective element.
  • the color beam splitter previously used there can be replaced by an active spectrally selective element, namely by a spectrally selective element which is suitable for hiding or displaying excitation light of different wavelengths or couple.
  • This spectrally selective element serves on the one hand for coupling the excitation light of at least one light source into the microscope and on the other hand for masking the excitation light scattered and reflected at the object or the corresponding excitation wavelength from the light coming from the object via the detection beam path.
  • the spectrally selective element has a dual function, with both functions being virtually positively coupled.
  • the spectrally selective element may be a passive element for facilitating the dual function discussed above.
  • the spectrally selective element could be designed as a transparent optical grating or as a holographic element. It is likewise conceivable to carry out the spectrally selective element as a passive AOD (Acousto-Optical-Deflecor) or as a passive AOTF (Acousto-Optical-Tunable-Filter).
  • the spectrally selective element is an active component, for example an acousto-optically and / or electro-optically operating element.
  • an AOD Acoustic-Optical-Deflector
  • an AOTF Acoustic-Optical-Tunable-Filter
  • an active spectrally selective element is used here, for example an AOD or an AOTF.
  • the object of these active components is to couple the excitation light of the light source or of the laser or the laser ⁇ ill1, ⁇ ill2 ⁇ ill3 ,..., ⁇ illn into the illumination beam path and thus into the microscope, and then by beam scanning to stimulate the fluorescence distribution in the object.
  • the fluorescent light coming from the object can pass through the active spectrally selective element almost undisturbed. In this case, the light scattered or reflected by the object is largely reflected out with the excitation wavelengths of the light source or the laser or the laser from the detection beam path.
  • the fluorescent light to be detected with a spectral distribution around the wavelengths ⁇ fluo1 , ⁇ fluo2 , ..., ⁇ fuon together with the scattered at the object or reflected excitation light with the wavelengths ⁇ ill1 , ⁇ ill2 , ..., ⁇ illn now passes through the AOD in the reverse direction.
  • the excitation light having the wavelengths ⁇ ill1 , ⁇ ill2 , ..., ⁇ illn is deflected out of the detection beam path toward the laser (1st order) because of the specific adjustment of the AOD.
  • the "spectrally remaining" fluorescent light can be detected in an improved manner by the wavelengths ⁇ fluo1 , ⁇ fluo2 ,..., ⁇ fluor compared to a conventional color beam splitter (0th order). This makes it possible in any case the adjustment of the coupling of different laser easier than in the prior art (there using conventional color beam splitter in a filter wheel) make.
  • a subsequent switching of further AOTFs could selectively regulate the individual wavelengths in their power after the beam merging.
  • an AOTF with corresponding frequencies ⁇ 1 , ⁇ 2 ,..., ⁇ n can be connected simultaneously, so that the different wavelengths vary in their excitation power and are optimized for the application.
  • the supply of the laser light can be effected by means of optical fiber.
  • the light source or the laser is coupled coaxially from the direction of the first order of the crystal.
  • the fluorescent light to be detected with a spectral distribution around the wavelengths ⁇ fluo1 , ⁇ fluo2 ,..., ⁇ fluon together with the excitation light scattered or reflected at the object with the wavelengths ⁇ fill1 , ⁇ fill2 ,..., ⁇ filln now pass through the AOTF in the opposite direction.
  • the excitation light having the wavelengths ⁇ fill1 , ⁇ fill2 ,..., ⁇ filln is deflected out of the detection beam path in the direction of the light source or the laser due to the specific setting of the OTF.
  • the "spectrally remaining" fluorescent light around the wavelengths ⁇ fluo1 , ⁇ fluo2 ,..., ⁇ fluon can be detected in an improved manner (0th order) compared to the conventional color beam splitter .
  • the fluorescence light will spectrally fan out after passing through the respective active element due to the dispersion that occurs.
  • the excitation pinhole is arranged, which is identical to the detection pinhole.
  • the property of the crystal, the light beam of the 0th order spectrally convincedfächem by the prism effect, is used for detection.
  • the dispersive element of the multiband detector is combined with the color beam splitter to form a component, whereby all other, the conventional detection beam path downstream and attributable to further losses in the fluorescence intensity color beam splitter omitted.
  • the technique discussed above can be used in combination with a wavelength tunable laser light source - e.g. Dye laser, OPO (optically parameterized oscillator).
  • Electron beam collision light source - enabling extremely flexible fluorescence microscopy applications.
  • the adjustment or control of the excitation wavelength can be coupled directly to the drive unit of one of the previously described spectrally selective elements, so that only this excitation wavelength coaxially coupled into the excitation beam path of the microscope and again only this wavelength is hidden from the detection beam path.
  • the coupling or forced coupling of the light source with the beam-splitting element can be done either manually or automatically or even according to a predefinable rule, this possibility being adapted to the respective requirement profile.
  • the excitation wavelength and the beam splitter can be changed in a suitable manner after each scanned image plane.
  • multi-color fluorescence objects can be detected.
  • a line-by-line switching is also conceivable.
  • Multi-Color FISH fluorescence in situ hybridization
  • different light sources can be used as long as they are suitable for fluorescence excitation.
  • a white light source a light source for using an optically parameterized oscillator, an electron beam collision light source or a laser light source come into question, wherein the laser light source can be variably tunable in wavelength.
  • Laser light sources with different wavelengths or a multi-laser light source is or are usable.
  • Fig. 1 documents the state of the art and shows a conventional optical arrangement in the beam path of a suitable for fluorescence excitation light source, which is an optical arrangement in the beam path of a confocal laser scanning microscope.
  • the laser scanner 1 is shown only symbolically.
  • a total of three lasers 2 are provided as light sources, which couple with their excitation light 3 via spectrally selective elements 4 into the illumination beam path 5 of the microscope.
  • the excitation light 3 is coupled into the illumination beam path 5 and arrives via a further mirror 8 as an excitation light 9 to the laser scanner 1.
  • the spectral selective elements 4 are a mirror 6 and a color beam splitter 7.
  • the light coming back from the object 10, which is also only symbolically represented-this is the excitation light 9 scattered and reflected on the object, on the one hand, and the fluorescent light 11 emitted by the object 10-passes through the mirror 8 to the spectrally selective element 4, where Here is the color beam divider 7. From there, the excitation light 9 or the excitation wavelength is masked out of the light 13 coming from the object 10 via the detection beam path 12 and returns to the lasers 2 as the returning excitation light 9.
  • the detection light 14, which is not deflected by the color beam splitter 7, arrives directly at the detector 15th
  • excitation light 3 of different wavelengths coming back through the spectrally selective element 4 can be masked out. This is especially in Fig. 3 shown.
  • the spectrally selective element 4 can be set to the excitation wavelength to be blanked out. This can be the embodiments of the Fig. 2 . 3 and 8th . 9 especially good.
  • the excitation light 3 may have different wavelengths.
  • the excitation light 3 passes through a mirror 6 and over additional optical element, namely via a lens 16 to an AOTF 17, which operates as a spectrally selective element. From there, the excitation light 3 again passes through an additional optical element - in the exemplary embodiment chosen here, a lens 18 - and via a mirror 8 to the laser scanner 1.
  • the returning light-reflected excitation light 9 and detection light 11 - passes through the mirror 8 and the lens 18 back into the AOTF 17 and is partially hidden there according to the wiring of the AOTF 17.
  • the detection light or fluorescent light 11 is guided via the detection beam path 12 to the detector 15 (0th order).
  • the returning excitation light 9 is guided back to the laser 2 via the lens 16 and the mirror 6 and is thus hidden from the detection beam path 12.
  • the light 13 coming from the object comprises fluorescent light 11 and returning excitation light 9, where the AOD 19 leads the returning fluorescent light as detection light 14 to the detector 15.
  • the returning excitation light 9 is hidden and passes through lenses 16 to the respective lasers 2.
  • Fig. 4 shown example which does not belong to the invention comprises as a spectrally selective element 4, a transparent grid 20, via the transparent grid 20 simultaneously three lasers 2 couple their excitation light 3 in the illumination beam path 5 of the microscope.
  • the transparent grid 20 blankes out the excitation light 9 coming back from the object 10 from the detection beam path, so that this light passes back to the lasers 2.
  • the fluorescent light 11 to be detected passes via the detection beam path 12 to the detector 15.
  • Fig. 5 shows the possibility of a dispersion correction, wherein the object returning light 13 in the AOTF 17 or AOD 19 passes. There, the returning detection light 14 is - compulsorily - spectrally fanned out and over downstream elements - AOD / AOTF - parallelized and finally converged. The spectrally combined detection light 14 passes from there to the in Fig. 5 not shown detector 15.
  • the light coming from the object 13 is fanned out by AOD 17 / AOTF 19, wherein the fanned detection light 14 via another passive spectrally selective element 4-AOTF 17 or AOD 19 - converted via a lens 21 with field correction and by a detection pinhole 22 or passes through a detection gap to the detector 15.
  • the spectrally selective element 4 is an AOTF 17 or an AOD 19, these elements comprise a special crystal with dispersion-free 0th order.
  • This crystal or this spectrally selective element is excited or acted upon via a piezoelectric element 23.
  • Fig. 7 shows particularly clearly that the light coming from the object 13 is split in the AOTF 17 and AOD 19, wherein the detection light 14 as a dispersion-free light 0th order passes through the crystal unhindered.
  • the excitation light 9 returning from the object is deflected as first order light and guided back to the lasers (not shown here).
  • Fig. 8 shows a special detection taking advantage of the spectral fanning of the spectrally selective element 4, wherein here in the concrete, an AOTF 17 is used.
  • the light 13 coming from the object 10 is spectrally split in the AOTF 17, whereby the detection light 14 passes via a lens 16 and a mirror 6 to a multiband detector 24 or spectrometer.
  • the mirror 6 leads to an extension of the distance, so that a fanning of the returning detection light 14 up to the multi-band detector 24 is favored.
  • the excitation light 9 hidden in the AOTF 17 passes back to the laser 2 via the lens 16 and the mirror 8.
  • Fig. 9 in a schematic representation of the embodiment Fig. 8 , where - in addition - in the detection beam path in front of the multiband detector 24, a variable gap filter 25 is arranged.
  • This slit filter 25 is arranged in the detection beam path 12 immediately in front of the detector 15 and can be positioned in the detection beam path.
  • the gap 26 of the slit filter 25 is variable, so that a spectral selection of the detection light 14 is also possible in this respect.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Optical Modulation, Optical Deflection, Nonlinear Optics, Optical Demodulation, Optical Logic Elements (AREA)
  • Spectrometry And Color Measurement (AREA)
  • Optical Elements Other Than Lenses (AREA)
  • Diffracting Gratings Or Hologram Optical Elements (AREA)

Claims (17)

  1. Microscope à balayage à laser confocal comportant un dispositif optique qui est disposé sur le chemin de faisceau d'une source lumineuse appropriée pour la stimulation de la fluorescence, et est disposée sur le chemin de faisceau du microscope à balayage à laser confocal, dans lequel le dispositif optique comprend au moins un élément spectralement sélectif (4) destiné à injecter la lumière de stimulation (3) d'au moins une source lumineuse (2) dans le microscope et à éliminer la lumière de stimulation (3) diffusée et réfléchie sur un objet (10) ou la longueur d'onde de stimulation de la lumière (13) provenant de l'objet (10) par l'intermédiaire du chemin du faisceau de détection (12), caractérisé en ce que l'élément spectralement sélectif (4) peut être réglé sur la longueur d'onde de stimulation devant être éliminée et en ce que l'élément spectralement sélectif (4) est un composant actif.
  2. Microscope à balayage à laser confocal selon la revendication 1, caractérisé en ce qu'une lumière de stimulation (3, 9) ayant des longueurs d'onde différentes peut être éliminée par l'élément spectralement sélectif (4).
  3. Microscope à balayage à laser confocal selon la revendication 1, caractérisé en ce que l'élément spectralement sélectif (4) fonctionne de manière acousto-optique et/ou électro-optique, ou en ce que l'élément spectralement sélectif (4) est réalisé sous la forme d'un AOD (Acousto-Optical Deflector, déflecteur acousto-optique) (19), ou en ce que l'élément spectralement sélectif (4) est réalisé sous la forme d'un AOTF (Acousto-Optical-Tunable-Filter, pour filtre acousto-optique accordable) (17).
  4. Microscope à balayage à laser confocal selon la revendication 3, dans lequel une source de lumière ayant des longueurs d'onde différentes peut être injectée, caractérisé en ce que l'AOTF (17) peut simultanément être câblé avec des fréquences correspondantes.
  5. Microscope à balayage à laser confocal selon l'une quelconque des revendications 1 à 4, caractérisé en ce que, pour une régulation spécifique de la puissance de longueurs d'ondes individuelles, au moins un autre élément actif ou spectralement sélectif est connecté en aval de l'élément spectralement sélectif (4).
  6. Microscope à balayage à laser confocal selon la revendication 5, caractérisé en ce que l'autre élément spectralement sélectif est un AOD (19) ou un AOTF (17).
  7. Microscope à balayage à laser confocal selon l'une quelconque des revendications 1 à 6, caractérisé en ce que la stimulation de la source lumineuse (2) par l'élément spectralement sélectif (4) s'effectue manuellement ou automatiquement ou conformément à une instruction pouvant être librement définie.
  8. Microscope à balayage à laser confocal selon l'une quelconque des revendications 1 à 7, caractérisé en ce qu'au moins un autre élément optique est connecté en amont et/ou en aval de l'élément spectralement sélectif (4).
  9. Microscope à balayage à laser confocal selon la revendication 8, caractérisé en ce qu'il est prévu en tant qu'autre élément optique un diviseur de faisceau coloré destiné à une décomposition spectrale supplémentaire.
  10. Microscope à balayage à laser confocal selon l'une quelconque des revendications 1 à 9, caractérisé en ce que des moyens destinés à effectuer une réflexion multiple et qui produisent un grossissement angulaire de la dispersion du faisceau de détection sont disposés sur le chemin du faisceau de détection (12).
  11. Microscope à balayage à laser confocal selon l'une quelconque des revendications 1 à 10, caractérisé en ce qu'un filtre séparateur (25) est disposé sur le chemin du faisceau de détection (12), de préférence immédiatement avant le détecteur (15).
  12. Microscope à balayage à laser confocal selon l'une quelconque des revendications 1 à 11, caractérisé en ce qu'un spectromètre destiné à détecter la dispersion spectrale est disposé sur le chemin du faisceau de détection (12) après l'élément spectralement actif (4).
  13. Microscope à balayage à laser confocal selon l'une quelconque des revendications 1 à 12, caractérisé en ce que les longueurs d'onde de stimulation éliminées sont déviées dans la direction des sources lumineuses (2) en dehors du chemin du faisceau de détection (12).
  14. Microscope à balayage à laser confocal selon l'une quelconque des revendications 1 à 13, caractérisé en ce que la source lumineuse (2) est réalisée sous la forme d'une source de lumière blanche, ou en ce que la source lumineuse (2) est réalisée sous la forme d'un oscillateur paramétrique optique (OPO), ou en ce que la source lumineuse (2) est réalisée sous la forme d'une source lumineuse à collision de faisceaux d'électrons, ou en ce que la source lumineuse (2) est réalisée sous la forme d'une source de lumière laser.
  15. Microscope à balayage à laser confocal selon la revendication 14, caractérisé en ce que la source lumineuse est accordable à des longueurs d'onde variables.
  16. Microscope à balayage à laser confocal selon la revendication 15, caractérisé en ce que la source de lumière laser comprend un laser ayant des longueurs d'onde différentes.
  17. Microscope à balayage à laser confocal selon l'une quelconque des revendications 1 à 16, caractérisé en ce que l'élément spectralement sélectif (4) est conçu de manière à ce qu'une dispersion spectrale de la lumière de détection (11) soit au moins évitée dans une large mesure.
EP99915483.4A 1998-02-19 1999-02-19 Systeme optique a element spectroselectif Expired - Lifetime EP1055144B1 (fr)

Applications Claiming Priority (3)

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DE19806867 1998-02-19
DE19806867 1998-02-19
PCT/DE1999/000459 WO1999042884A1 (fr) 1998-02-19 1999-02-19 Systeme optique a element spectroselectif

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EP1055144A1 EP1055144A1 (fr) 2000-11-29
EP1055144B1 true EP1055144B1 (fr) 2015-01-14

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EP (1) EP1055144B1 (fr)
JP (2) JP4435977B2 (fr)
WO (1) WO1999042884A1 (fr)

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JP4435977B2 (ja) 2010-03-24
JP2002504708A (ja) 2002-02-12
EP1055144A1 (fr) 2000-11-29
US20030133189A1 (en) 2003-07-17
WO1999042884A1 (fr) 1999-08-26
US6654165B2 (en) 2003-11-25
US6510001B1 (en) 2003-01-21
JP2009139969A (ja) 2009-06-25

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